Understanding the genetic basis of complex traits is critical for advancing medicine, evolutionary biology, and agriculture. A central problem limiting progress in this area is that genetic variants can interact in complicated ways, making their effects difficult to detect in standard genetic mapping studies. In particular, recent research suggests that 'higher-order' genetic interactions involving three or more loci commonly contribute to trait variation. Little is known about these complex genetic effects that appear to b surprisingly important. Determining how higher-order interactions arise at the molecular level and contribute to traits may prove vital to solving the 'missing heritability' problem in humans and other species. Our goal in this proposal is to fully characterize the molecular bases and phenotypic effects of multiple higher-order interactions. We have identified three traits in Saccharomyces cerevisiae crosses that are specified by sets of interacting loci and can each provide unique insights into how higher-order interactions contribute to phenotypic variation. We will comprehensively dissect a discrete morphological trait (Aim 1), an environmentally sensitive growth defect (Aim 2), and a quantitative drug sensitivity phenotype (Aim 3) using the power of the yeast model system. By completing our work, we will provide some of the first direct, mechanistic insights into higher-order interactions. Relevance: Elucidating the connection between genotype and phenotype is critical to achieving new heights in medicine, evolutionary biology, and agriculture. However, for many phenotypes of interest, genome-wide association and linkage studies have only identified a small fraction of the traits' genetic bases. The missing heritability that remains in these studies is arguably the primary barrier to a new era of personalized medicine and genome sequence-informed biology. Higher-order genetic interactions involving three or more loci could represent a significant source of missing heritability, as they may not be detected in standard genetic mapping studies. We are among a small number of research groups that have found compelling evidence that higher-order interactions can result in major, unexpected phenotypic effects. In this proposal, our goal is to understand the functional mechanisms that underlie higher-order interactions. Completion of our work should provide crucial insights into the potential contribution of higher-order interactions t the missing heritability of complex traits in humans and other species.